Detection of CNS disease

ABSTRACT

Techniques for the detection of disease of the central nervous system,  pacularly of bovine spongiform encephalopathy or &#34;mad cow disease&#34;, scrapie and related states, are provided based upon the detection of a factor present in the urine of affected animals or humans. That factor is defined in terms of elution data from an ion exchange column and by electrochemical characteristics.

The present invention relates to techniques for the detection of diseaseof the central nervous system, particularly bovine spongiformencephalopathy, scrapie, Jacob-Creuzfeld syndrome and related states.

It has been noted that serotonin levels are reduced in the blood ofscrapie infected sheep (Chatelain et al (1984) CR Soc. Biol. 178,664-670, and that some electrochemical changes occur in the urine ofsuch sheep (Combrisson et al (1991) Bull. Acad. Vet. de France 64,257-266; Banissi-Sabourdy et al (1992) Bioelectrochem & Bioenergetics28, 127-147, but no reliable test has resulted from these findings.

At present there is no diagnostic test available for the screening ofanimals, including humans, for spongiform encephalopathy type diseaseand the confirmation of its presence is usually by direct observation oftissues in autopsy. There remains a particular need for a method ofscreening live animals, particularly prior to slaughter for consumptionpurposes, and preferably before their milk is consumed, and mostpreferably for a non-invasive test. The present inventor has nowidentified a factor present in the biological fluids of affectedanimals, particularly their urine, the amount of which correlates wellwith the occurence of these diseases.

The present invention provides a method for determining the presence ofcentral nervous system disease in a human or animal body by analysis ofone or more of its biological fluids characterised in that the fluid isanalysed for the presence of a factor associated with a fractionobtainable from an ethanol extract of urine from animals having thedisease, that fraction being elutable from a 0.2M Tris buffer pH 7.0equilibrated DEAE-Sepharose Cl ion exchange column (particularly a CL6B)increasing gradient elution using from 0 to 0.2M sodium chloride in0.02M Tris buffer pH 7.0; the fraction being elutable with between 0.08Mand 0.015M sodium chloride, particularly between 0.08M and 0.09M sodiumchloride in Tris buffer pH 7.0; wherein the amount of this factor isrelated to presence of the disease state.

The most convenient method for detecting this `disease` factor may ofcourse vary depending upon the fluid to be tested, eg. whether CSFfluid, whole blood or a fraction thereof, or urine. The sampling ofurine renders the test suitable for routine screening byelectro-chemical detection means and can avoid the need for invasivemethods. Other tests, eg. colorimetric and immunological, will also beapplicable.

A preferred aspect of the present invention relates the amount of the`disease factor` with the amount of one or more reference factorspresent in the fluid, particularly in urine, and uses the ratio of theseto indicate disease presence. These reference factors are a referencefactor (2) that is associated with a fraction elutable with between 0.09and 0.13M sodium chloride, and a reference factor (3) associated with afraction elutable with between 0.13 and 0.2M sodium chloride; both usingthe ion-exchange column technique described above and the same ethanolextract of disease animal's urine.

The second reference factor (3) has been found to be uric acid, thefirst reference factor (2) has been found to have properties associatedwith tyrosine or a precursor or derivative thereof having at least one,and possibly two or three, phenolic groups including one phenol grouppara to a substitute side chain. It should be realised that the amountof material isolatable from urine is very small and has so far provendifficult to identify.

The disease factor (1) has been found to have properties consistent withhaving a substituted phenyl or histidine nucleus, also with apara-hydroxy substituent and at least one methyl substituent.

Both disease factor (1) and reference factor (2) have similar physicalproperties. Both are water soluble and soluble in methanol and ethanol,both are bound to aminopropyl solid phase extraction columns but onlyreference factor (2) can be desorbed using 0.5M HCl in 50:50methanol/water; thus in this way reference factor can be separated fromdisease factor. The disease factor does not come off this column ineither 100% methanol or 1:1 methanol:5M HCl in water. Both referencefactor (2) and disease factor (1) are bound to anion exchange materialsuch as DEAE-Sepharose, being eluted with between 0.08 to 0.15M sodiumchloride, the reference factor being desorbed slightly after the diseasefactor, ie. above 0.09M as described above. The reference factor (2) maybe further identified by its Rf factor on TLC RP18 plates using an 80:20water:methanol solvent.

It has been found that measurement of the difference in height of anelectrochemically measured peak obtained relating to the amount ofreference factor (2) and one obtained relating to the disease factor,divided by the difference in height of peaks relating to the referencefactors (2) and (3), provides a value indicative of disease state.Initial investigation shows that values of 1.0 or more correlate withabsence of disease while less than 1.0 correlates with disease status.It will be appreciated that any method that measures the relativeconcentrations of disease factor and one or both of these referencefactors may be used to indicate presence of the aforesaid diseases.Furthermore, it will be realised that in some disease formats, thedisease factor may occur in an altered form while still retaining itsbasic chemical characteristics; eg. as a conjugate or metabolite. Suchvariations of the factor will also be targetable for the purposes ofdetection and thus the present invention relates to their targeting inso far as they are so related and correlate to the diseases of interest.

In a further aspect of the present invention there is provided a testkit for determining the presence of central nervous system diseasecomprising one or more reagents capable of specifically detecting thedisease factor described above.

Most conveniently these agents will be specific binding assay agentscapable of specific detection of the approximate amount of the diseaseagent in a biological fluid. Typically such agents will be antibodiesraised to immunogens formed from the purified fraction obtained by itselution from a column, eg using 0.08 to 0.09M NaOH in 0.02M Tris bufferfrom a DEAE column as described above. Such antibodies may be monoclonalantibodies or polyclonal antibodies raised in the known manner usingsuitably selectable immunogen design, eg ovalbumen conjugate, andadjuvant if necessary as will be determined by simple benchexperimentation; the antibodies being conventionally labelled fordetection of disease agent in the known manner. Alternatively the testreagents may include specific chemical test agents for the agent.

Isolation of the fraction containing the disease factor and theapplication of electrochemical techniques to the method of the inventionwill be illustrated by way of the non-limiting examples and relatedFigures provided below.

FIGURES

FIG. 1: shows a voltammogram of a sample derived from BSE positive urineas obtained using the method of Example 1.

FIG. 2: shows a voltammogram of a sample derived from BSE negative urineas obtained using the method of Example 1.

EXAMPLE 1

Urine samples were taken from animals (cattle) under investigation andscreened using a commercial electrode cell (ESA Analytical: CoulochemII, 5010 electrode assembly) containing three porous carbon electrodes,these being a reference electrode, a working electrode and a secondaryelectrode. The sample was prepared by mixing 200 μl of urine with 768 μldeionised water and 32 μl hydrochloric acid (10.8N) (or concentratedsulphuric acid) in order to acidify it prior to introduction into theelectrode cell. The cell was operated using the following electricalparameters:

Applied potential cycles:

(i) 175 mV to 1325 mV

(ii) 1325 mV to 175 mV

These cycles were repeated one or more times and the voltage change rateused was usually 10 mV/second, although any rate in the range eg. 1-100mV/second was found to be usable. The induced current was measured at 20μamps full scale deflection but 2-200 μamps f.s.d. may be convenientlyused. The output of the cell electrodes was transcribed to a X-Y linearplotter.

Results

Three peaks were produced on the plotter output;

(a) Primary Peak 1: occurs only on the second and subsequent cyclicsweeps during increasing applied voltage between 650 and 850 mV (mean at750 mV); this being referred to also as disease correlating peak.

(b) Reference Peak 2: occurs only on the second and subsequent cyclicsweep during increasing applied voltage at approximately 100 mV lowerthan the Primary Peak 1.

(c) Reference Peak 3: occurs only on the first cyclic sweep duringincreasing applied voltage at approximately 25 to 100 mV higher than thePrimary Peak 1, this being resultant from irreversible oxidation andbeing found to correspond to uric acid.

Quantitation

The Primary Peak 1 is found to be increased in the diseased state butbecause urine from different animals, whether captured mid-flow, bycannula or from post-mortem bladders, varies considerably inconcentration/dilution. Reference peaks 2 and 3 were used to normalisereadings by serving as internal standards relating to urine dilutionwithin the sample.

Thus the difference in height of the Primary Peak 1 and Reference Peak 2divided by the difference in height of the Reference Peaks 2 and 3,provides a determining numerical value indicative of disease state:

Peak 2-Peak 1/Peak 3-Peak 2 greater than or equal to 1.0 --SampleNegative

Peak 2-Peak 1/Peak 3-Peak 2 less than 1.0--Sample Positive

Assessment of Test

A trial was run using blind samples analysed by the test as describedabove and the results compared to histopathological diagnosis results,the results of which are provided in Table 1 wherein positiveidentifications of disease state are indicated by + and negativeidentifications by -.

                  TABLE 1                                                         ______________________________________                                        Sample  Urine    Histol  Sample  Urine                                                                              Histol                                  ______________________________________                                        1       +        +       10      -    -                                       2       -        -       11      +    +                                       3       +        +       12      +    +                                       4       +/-      +       13      +    +                                       5       +        +       14      +    +                                       6       -        -       15      +    +                                       7       +        +       16      +    +                                       8       +        +       17      +    +                                       9       +        +       18      +    +                                       ______________________________________                                    

EXAMPLE 2

Urine from diseased cattle was subjected to a number of isolationprocedures to provide purified fractions containing the identifieddisease correlating factor. One preferred procedure is as follows:

(a) Ethanol extraction: Inorganic salts and ethanol insoluble compoundswere precipitated from urine by addition of 4×volume of absolute ethanoland filtering off the precipitate. The filtrate was evaporated todryness and the compounds of interest redissolved in ethanol, evaporatedto dryness again and taken up in 0.02M Tris buffer at pH 7.0 for aseparation stage (b).

(b) The sample from step (a), obtained from 50 ml of urine, was absorbedonto a DEAE-Sepharose CL6B (Pharmacia) ion exchange column previouslyequilibrated with 0.2M Tris buffer pH 7.0. The column was washed clearof non-adsorbing material and subjected to an increasing gradientelution from 0 to 0.2M sodium chloride in 0.02M Tris buffer pH 7.0.

4 ml fractions were collected and assessed for activity on theelectrochemical detector. Peak 1 was eluted between 0.08 and 0.09Msodium chloride and found to be essentially electrochemically pure. Peak2 was eluted at between 0.09 and 0.13M sodium chloride and was alsofound to be electrochemically pure. Peak 3 was eluted at between 0.13and 0.2M sodium chloride. The exact elution salt concentration requiredappears to depend on the amount of original urine and its originalconcentration.

We claim:
 1. A method for determining the presence of a transmissiblespongiform encephalopathy in a nonhuman animal wherein its urine isanalyzed for the presence of a disease factor (1) an increase in theamount of which is related to presence of the disease state, whereinsaid disease factor (1);(a) has properties consistent with its having asubstituted phenyl or histidine nucleus, (b) is associated with afraction obtainable from an ethanol extract of urine obtained fromanimals having Bovine spongiform encephalopathy, that fraction beingelutable from a 0.2M Tris buffer pH 7.0-equilibrated DEAE-Sepharose Clion exchange column by increasing gradient elution from 0.08 and 0.09Msodium chloride in 0.02M Tris buffer pH 7.0, (c) is detectable in thatfraction by means of its electrochemical properties, and has mid-pointpotential of between 650 and 850 mV, (d) is soluble in water, methanoland ethanol, and (e) can be bound to aminopropyl solid phase extractioncolumns, but is not recoverable from said column using 100% methanol or5M aqueous HCl/methanol 1:1 mixture as eluant.
 2. A method as claimed inclaim 1 wherein the disease factor (1) has a para-hydroxy substituentand at least one methyl substituent.
 3. A method as claimed in claim 1wherein the amount of the disease factor (1) is compared with the amountof a reference factor (2) that:(a) is associated with an ethanolextractable urine fraction, that fraction being elutable from a 0.2MTris buffer pH 7.0-equilibrated DEAE-Sepharose Cl ion exchange column byincreasing gradient elution using from 0.09 and 0.13M sodium chloride in0.02M Tris buffer pH 7.0, (b) is detectable in that fraction by means ofits electrochemical properties, and has mid-point potentialapproximately 100 mV lower than that of the disease factor (1), and (c)is soluble in water, methanol and ethanol, and can be bound to anaminopropyl solid phase extraction column, but is recoverable from saidcolumn using 0.5M HCl in 50:50 methanol/water.
 4. A method as claimed inclaim 3 wherein reference factor (2) has properties associated withtyrosine or a precursor or derivative thereof having at least onephenolic group.
 5. A method as claimed in claim 3 wherein referencefactor (2) includes one phenol group para to a substitute side chain. 6.A method as claimed in claim 3 wherein the disease factor (1) iselectrochemically measured.
 7. A method as claimed in claim 3 whereinthe amount of the disease factor is compared with the amount of areference factor (3) that is uric acid.
 8. A method as claimed in claim7, wherein the disease factor (1), reference factor (2) and referencefactor (3) are electrochemically measured and the difference in heightof peak obtained relating to the amount of reference factor (2) and thatobtained relating to the disease factor (1), is divided by thedifference in height of peaks relating to the reference factors (3) and(2), and the value obtained is used to determine disease state.
 9. Amethod as claimed in claim 8 wherein the disease factor (1), referencefactor (2) and reference factor (3) are eluted from an ion exchangecolumn prior to electrochemical measurement.
 10. A method as claimed inclaim 9 wherein the ion exchange column contains DEAE-Sepharose CL6B.11. A method of generating antibodies for use in a method of diagnosisof a transmissible spongiform encephalopathy in a nonhuman animal,comprising:(a) purifying the disease factor (1) of claim 1 from thefraction obtainable from an ethanol extract of urine obtained formanimals having Bovine spongiform encephalopathy, that fraction beingelutable from a 0.2M Tris buffer pH 7.0 equilibrated DEAE-Sepharose Clion exchange column by increase gradient elution from 0.08 and 0.09sodium chloride in 0.02M Tris buffer pH, (b) administering the diseasefactor (1) to an animal in the form of an immunogen conjugate, and (c)recovering antibodies from that animal.